American mitogenome reference for the tropical brown dog tick, Rhipicephalus linnaei (Audouin, 1826)

The brown dog tick, Rhipicephalus linnaei (Audouin, 1826), is distributed across the American continent and is formerly known as the “tropical lineage”. It belongs to the Rhipicephalus sanguineus (Latreille, 1806) species complex, referred to as R. sanguineus (sensu lato). Mitochondrial genome sequences are frequently used for the identification and represent reference material for field studies. In the present study, the entire mitochondrial genomes of R. linnaei (∼15 kb) collected from dogs in Mexico were sequenced and compared with available mitogenomes of R. sanguineus (s.l.). The mitochondrial genome is ∼90% identical to the reference genome of R. sanguineus (sensu stricto, former “temperate lineage”) and > 99% identical to R. linnaei mitogenome derived from the neotype. Two additional mitogenomes were obtained and described as R. linnaei and R. turanicus from dogs in Saudi Arabia. The present study delivers a molecular reference for R. linnaei from America and complements R. linnaei mitogenomes from Africa, Asia and Australia. We propose to consider the complete mitogenome, as the reference for American R. linnaei, even when partial mitochondrial cox1, 12S rRNA or 16S rRNA genes are characterised.


Introduction
The brown dog tick Rhipicephalus sanguineus (sensu lato) is a wellrecognised tick parasitising dogs in the Americas (Dantas-Torres, 2010).Early studies investigating the distribution and genetic diversity laid the foundation for recognition of "southern" and "northern" lineages in South America (Moraes-Filho et al., 2011).The "southern" lineage was later recognised as the "temperate lineage" with the formal name Rhipicephalus sanguineus (Latreille, 1806) referred here to as R. sanguineus (sensu stricto) via a redescription of a neotype (Nava et al., 2018).The "northern" lineage or the "tropical lineage" was recently redescribed as Rhipicephalus linnaei (Audouin, 1826) using a neotype from Egypt ( Šlapeta et al., 2022).Moraes-Filho et al. (2011) have genetically analysed brown dog ticks from Linares, Nuevo Leon State, Mexico, and used partial ~460 bp fragment of the mitochondrial 16S rRNA gene to assign them to the "northern" lineage or what is now recognised as R. linnaei.More recently, brown dog ticks were demonstrated on dogs and a single cat infested by nymphs of the tick species in the State of Puebla in Mexico, and molecularly identified as R. linnaei based on partial mitochondrial 16S rRNA gene (Salceda-Sánchez et al., 2023).Finally, the presence of R. linnaei was demonstrated across 12 states in Mexico based on partial fragments of the mitochondrial 12S rRNA and 16S rRNA genes and morphological investigation (Almazán et al., 2023).The presence of R. linnaei was further demonstrated across the state of Morelos in Mexico using a partial fragment of the mitochondrial 16S rRNA gene (Nieto-Cabrales et al., 2024).In North America, in particular the USA, both species R. sanguineus (s.s.) and R. linnaei are present (Jones et al., 2017).These two species predominate in distinct, overlapping geographies and their activity peaks at different times of the year (Grant et al., 2023).In Canada, brown dog ticks are only rarely detected most often linked to international travel (Myers et al., 2024).All of the North American studies utilised partial 340-370 bp fragment of the mitochondrial 12S rRNA gene to determine the identity of Rhipicephalus species (Jones et al., 2017;Grant et al., 2023;Myers et al., 2024).Based on these results, the brown dog tick in Mexico can be considered to represent only R. linnaei and R. sanguineus (s.s.) is currently absent.
Despite the interest on the brown dog ticks, R. sanguineus (s.l.), and general acceptance of species (former lineages) identity determination based on partial mitochondrial sequences, there is currently no reference of a complete mitochondrial genome (mtDNA) also referred as 'mitogenome' from American specimen of R. linnaei.Mitogenomes enable comparison of the entire genome (~14 kb) compared to small fragments (300-500 bp) and play a crucial role when comparing tick species and establishing robust taxonomy coupled with a morphological investigation (Burger et al., 2014;Kneubehl et al., 2022;Šlapeta et al., 2022, 2023;Barker et al., 2023).
This study aimed to provide a reference mitogenome sequence for the brown dog tick R. linnaei from America.To do so, ticks were collected from dogs in Mexico, whole genome sequencing was performed and complete mitogenomes of R. linnaei assembled.The assembled mitogenomes were compared to available mitogenomes of R. sanguineus (s.l.).The sequence analysis was enriched by the addition of new Rhipicephalus spp.from the Arabian Peninsula.Overall, this study demonstrates the presence of R. linnaei in the Americas, Asia, Africa, and Australia based on their complete mitogenomes.

Available material and morphological identification
Ticks were collected from dogs in northern Mexico in 2023 (Ciudad Victoria, Tamaulipas State;23.735000, − 99.130833).In total, 10 adult ticks (5 males and 5 females) from Mexico were used; all representing the genus Rhipicephalus.In addition, 10 ticks from dogs from Eastern Province of Saudi Arabia were collected in 2019 25.383333,49.600000);seven ticks represented the genus Rhipicephalus, and three were Hyalomma spp. that were not processed further.Tick specimens were stored in individual tubes with 70% (v/w) ethanol.All ticks were observed under a stereo microscope (SMZ-2B, Nikon, Australia) and a digital microscope (VHX-6000, KEYENCE Inc., Japan), and identified using published keys and guides (Walker et al., 2000).

DNA isolation and amplification of the partial mitochondrial cox1 gene
DNA was isolated from ticks stored in 70% (v/w) ethanol as previously described by Šlapeta et al. (2022).Briefly, dried ticks with incisions in their idiosoma were subjected to total tick genomic DNA (gDNA) isolation with the Monarch Genomic DNA Purification Kit (New England Biolabs, Melbourne, Australia).Exoskeletons were retained and preserved in 70% (v/w) ethanol.Extracted gDNA was stored at − 20 • C.
The ~600 nucleotide (nt) fragment of the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit 1 (cox1) gene was amplified using the primer set S0725/S0726, as described in Chandra et al. (2019) and Šlapeta et al. (2022).MyTaq™ Red Mix (Bioline, Meridian Bioscience, Melbourne, Australia) was used for DNA amplifications in 30 μl reactions, with 2 μl (approximately 0.5-10 ng) of template gDNA.All reactions included PCR-grade water (ddH 2 O) as a no-template control.The PCR reactions were performed in a T100™ Thermal Cycler (BioRad, South Granville, Australia), and the PCR products were sequenced at Macrogen Ltd. (South Korea).

Genome skimming of Rhipicephalus spp. and assembly of mitogenome from next-generation sequence data
The gDNA isolated from four adult Rhipicephalus spp.ticks was used for genome skimming via next-generation sequencing (NGS) using a NEBNext® DNA Library Prep Kit followed by NGS using 150 bp pairedend Illumina NovaSeq 6000 sequencing system at a depth of 3 Gb or 1 Gb of raw sequence data (Novogene, Singapore).To enrich our Rhipicephalus spp.data, published next-generation data from Colombia were used (Paez-Triana et al., 2023); the raw FastQ data for representative samples from two localities were retrieved from the project number PRJEB64029 at Sequence Read Archive (SRA) data in GenBank (htt ps://www.ncbi.nlm.nih.gov/sra).

Mitogenome phylogenetic analysis
Evolutionary analysis by maximum likelihood (ML) and minimum evolution method (ME).This analysis involved 30 nucleotide sequences.There was a total of 15,029 positions in the final dataset.Evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021).The evolutionary model was selected based on fitting 24 different nucleotide substitution models; the model with the lowest Bayesian Information Criterion (BIC) scores was considered to describe the substitution pattern.For the entire Rhipicephalus spp.mitogenome alignment, the best model was GTR (General Time Reversible model) + G (Gamma distribution) + I (model allowed for some sites to be evolutionarily invariable) with 67 parameters and BIC score 114032.785(ln = − 56580.937)and was used for ML analysis.For the alignment with R. linnaei mitogenomes, the best model was HKY (Hasegawa-Kishono-Yano model) + G (Gamma distribution) with 42 parameters and BIC score 57130.890(ln = − 28301.006)and was used for ML analysis.The ME evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004).Bootstrap test was used to calculate the percentage of replicate trees in which the associated mitogenomes clustered together (1000 replicates for ME, 100 replicates for ML).

Results
Ticks from Mexico morphologically belonged to R. sanguineus (s.l.) and exhibited features congruent with R. linnaei ( Šlapeta et al., 2022).Four ticks underwent DNA isolation with DNA concentration from 10.08 ng/μl to 0.42 ng/μl.Initial amplification of cox1 yielded sequences identical with those of R. linnaei and two of the DNA samples were processed for next-generation whole genome sequencing.Sequencing of DNA from the tick voucher JS6619 (A2) produced 25,273, 822 raw 150 nt reads totalling 3.8 Gb of raw data (Q30 = 92.71%;GC = 47.30%).Sequencing of DNA from the tick voucher JS6621 (B2) produced 31,876,342 raw 150 nt reads totalling 4.8 Gb of raw data (Q30 = 92.58%;GC = 47.30%).The assembly of the 150 bp pair reads, yielded 14,710 bp (#JS6619) and 14,711 (#JS6621) bp long contigs that were complete and represented circular mtDNA.The overlapping mtDNA regions using both libraries were almost identical except for 7 single nucleotide differences and one gap.The circular mtDNA of R. linnaei from Mexico encodes 13 protein-coding genes, two rRNA genes and tRNAs.The new mitogenomes were 99.9% identical with 20 and nucleotide differences from a reference R. linnaei mitogenome (NC_060409).The previously obtained partial sequences for the mitochondrial 16S rRNA gene (OR388555) by Nieto-Cabrales et al. ( 2024) from Mexican R. linnaei were 100% identical with the complete mtDNA obtained in this study as well as the reference mitogenome of R. linnaei (NC_060409).Similarly, mitochondrial 16S rRNA gene sequence (GU553075) from Moraes-Filho et al. (2011) that represents the original "northern" lineage is 100% identical with the newly obtained mitogenome data for the 16S rRNA gene.
Seven ticks from Saudi Arabia morphologically fitted the general description of R. sanguineus (s.l.) with some exhibiting features Fig. 1.Phylogenetic relationships of Rhipicephalus sanguineus (sensu lato) based on complete mitogenomes.A Complete 30 mitogenomes of R. sanguineus (s.l.) were aligned and the tree was inferred from nucleotide data; there were a total of 15,029 positions in the final dataset.The optimal tree shown was inferred using the Minimum Evolution (ME) method.The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.For the ME tree, all ambiguous positions were removed for each sequence pair (pairwise deletion option).The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates for ME/100 replicates for ML [maximum likelihood]) are shown next to the branches.The species from which the mitogenome belongs to is indicated on the right and colour coded.B Complete 18 mitogenomes of R. linnaei were aligned together with two R. camicasi mitogenomes as outgroup.The tree was inferred from nucleotide data, there were a total of 14,755 positions in the final dataset.The optimal tree shown was inferred using the Minimum Evolution (ME) method.The tree was inferred as described above.Each mitogenome is identified by its GenBank accession number and voucher identifier together with the country where it was collected.The new mitogenomes from this study are shaded.
We have then generated mitogenomes from three randomly selected FastQ libraries from tick collected on dogs from Santander (n = 2; ERR12084987 and ERR12085080) and Casanare (n = 1; ERR12085003), Colombia.The assembly from the 150 bp pair reads, yielded 14,714 to 14,716 bp long contigs that represented circular mtDNAs encoding 13 protein-coding genes, two rRNA genes and 22 tRNAs.Appending the Colombian brown dog tick mitogenomes to the Rhipicephalus spp.alignment and inferring their phylogenetic position demonstrated that they unambiguously belong to the R. linnaei clade of mitogenomes with > 99.8% nucleotide sequence identity to reference R. linnaei as well as the Mexican reference mitogenomes (Fig. 1B).

Discussion
Brown dog ticks are the most common ectoparasite of domestic dogs in Mexico (Tinoco-Gracia et al., 2009), where its presence was documented over 100 years ago (Macías-Valadez, 1923).The brown dog tick was associated with a rickettsiosis outbreak that occurred in northern Mexico in 1947( Álvarez-Hernández et al., 2017).Otherwise, infestations by the brown dog ticks were underestimated until 21st Century when outbreaks of rickettsiosis in the southwestern US state of Arizona and rural and suburban areas of Baja California in northwestern Mexico occurred in which the dog tick was demonstrated to be the vector (Demma et al., 2005;Parola et al., 2013).
The brown dog ticks, R. linnaei, identified in this study were collected from dogs in Ciudad Victoria, Tamaulipas State, in northern Mexico, where brown dog ticks are seen throughout the year in untreated animals.Using these ticks, we have provided the reference mitogenome for the brown dog tick R. linnaei from the American continent complementing those from Asia, Africa and Australia (Jia et al., 2020;Šlapeta et al., 2021, 2022) (Fig. 2).Generating mitogenomes of ticks and other parasites is possible using genome skimming via next-generation sequencing (Jia et al., 2020;Šlapeta et al., 2021;Papaiakovou et al., 2023).The cost of genome skimming for ticks is comparable to the cost of PCR amplification and bi-directional Sanger sequencing of three or more amplicons, yet it generates all mitochondrial full coding sequences as well as both 12S and 16S rDNA gene sequences compared to a partial amplicon (Kneubehl et al., 2022;Šlapeta et al., 2022, 2023).Besides mitogenome, genome skimming enables screening for pathogens vectored by these ticks including bacteria, protozoans, fungi, nematodes, and viruses (Jia et al., 2020;Paez-Triana et al., 2023).2023) generated shotgun next-generation sequence data from brown dog tick in Colombia that they considered the "tropical lineage" of R. sanguineus (s.l.) ticks in Colombia.Their focus was metagenomic identification of pathogens carried by these ticks, identifying Anaplasma phagocytophilum, Francisella tularensis, Theileria equi and the ubiquitous endosymbiont Coxiella mudrowiae (Paez-Triana et al., 2023).Here, we successfully used their data to complement our mitogenome from South-Central American countries for R. linnaei.The identity of R. linnaei from the Americas is now confirmed using complete mitogenomes from Mexico and Colombia.
During last century, literature referred to brown dog tick in Mexico as "R.sanguineus".However, two genetic lineages of R. sanguineus in the American continent were recognised (Moraes-Filho et al., 2011), "temperate" or "southern" lineage now representing the R. sanguineus (s.s.), and "tropical" or "northern" lineage now representing R. linnaei.The distinction was supported with partial mitochondrial 12S rRNA gene and 16S rRNA gene sequences (Moraes-Filho et al., 2015;Grant et al., 2023).Aside from morphology and genetics, biological differences between R. sanguineus (s.s.) and R. linnaei in America exist.For instance, R. sanguineus (s.s.) undergo diapause, which helps it to survive in temperate regions of Argentina, Brazil, Uruguay, and the USA, whereas in R. linnaei diapause does not occur, thus explaining why R. linnaei is found parasitising dogs throughout the year in South-Central American countries (Labruna et al., 2017).This is in line with the findings of Galindo-Velasco et al. ( 2020) who studied the annual infestation pattern of R. sanguineus (s.l.) in the State of Colima in Mexico, finding that infestation occurs throughout the year with similar infestations from spring to winter.
The distribution of R. linnaei in Mexico was documented by Villarreal et al. (2018), reporting it from the southern Mexican State of Oaxaca through southern California and western Arizona in the USA.While the Villarreal et al. (2018) study was based on partial mtDNA sequences, these sequences match with R. linnaei.Therefore, morphological studies combined with molecular analysis demonstrate that R. linnaei is widely distributed in Mexico (Almazán et al., 2023;Nieto-Cabrales et al., 2024).

Conclusions
The species complex R. sanguineus is recognised to harbour multiple species; of these, two are known to be established in the Americas, R. sanguineus (s.s.) from South America and North America and R. linnaei that is the only species in Central America but extends to the warmer climates in South and North America as well (Jones et al., 2017;Díaz et al., 2018;Myers et al., 2024).Despite the reclassification of the "tropical lineage" of R. sanguineus (s.l.) as R. linnaei, this tick is still referred to as the "tropical lineage".New mitogenome data from Mexico confirm that R. linnaei found in the tropics of the American continent is conspecific with the R. linnaei reference from Egypt.Based on these results, we conclude that tropical brown dog ticks from Mexico correspond to R. linnaei, making the term "tropical lineage" of R. sanguineus (s.l.) unnecessary in the Americas.Therefore, we propose using the complete mitogenome as the reference for American R. linnaei, even when only partial mitochondrial cox1, 12S rRNA or 16S rRNA genes are characterised.

Fig. 2 .
Fig. 2. World map distribution of Rhipicephalus sanguineus (sensu lato) based on complete mitogenomes.Each mitogenome is colour coded (as in Fig. 1) based on species identification, each circle represents one mitogenome.Reference sequences for R. linnaei are now available from the Americas, Africa, Asia and Australia.